EN ISO 16140:2003

Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods (ISO 16140:2003)

ISO 16140:2003 defines the general principle and the technical protocol for the validation of alternative methods in the field of microbiological analysis of food, animal feeding stuffs and environmental and veterinary samples for the validation of alternative methods which can be used in particular in the framework of the official control, and the international acceptance of the results obtained by the alternative method.
It also establishes the general principles of certification of these alternative methods, based on the validation protocol defined in ISO 16140:2003.
Where an alternative method is used on a routine basis for internal laboratory use without the requirement to meet (higher) external criteria of quality assurance, a less stringent comparative validation of the alternative method than that set in ISO 16140:2003 may be appropriate.

Mikrobiologie von Lebensmitteln und Futtermitteln - Arbeitsvorschrift für die Validierung alternativer Verfahren (ISO 16140:2003)

Diese Norm EN ISO 16140 legt das allgemeine Prinzip und die technische Arbeitsvorschrift für die Validierung alternativer Verfahren auf dem Gebiet der mikrobiologischen Untersuchung von Lebensmitteln und Futtermitteln sowie von Umwelt- und veterinärmedizinischen Proben (siehe 5.1.1.2.1) für folgende Punkte fest:
- Validierung von alternativen Verfahren, die besonders im Rahmen der amtlichen Lebensmittelüberwachung angewendet werden können;
- internationale Anerkennung der durch das alternative Verfahren ermittelten Ergebnisse.
Sie enthält auch die allgemeinen Prinzipien für die Zertifizierung dieser alternativen Verfahren, die auf der in der vorliegenden Norm EN ISO 16140 festgelegten Arbeitsvorschrift für die Validierung (siehe 4.2) beruhen.
Wenn ein alternatives Verfahren als Routineuntersuchung für die hausinterne Verwendung ohne die Anforderung, die (höheren) externen Kriterien der Qualitätssicherung zu erfüllen, angewendet wird, kann eine weniger strenge vergleichende Validierung des alternativen Verfahrens zweckmäßig sein als die, die in dieser Norm festgelegt ist.

Microbiologie des aliments - Protocole pour la validation des méthodes alternatives (ISO 16140:2003)

L'ISO 16140:2003 établit le principe général ainsi que le protocole technique de validation des méthodes alternatives dans le domaine de l'analyse microbiologique des aliments, des produits alimentaires pour animaux et des échantillons environnementaux et vétérinaires, en vue de la validation des méthodes alternatives pouvant être utilisées en particulier dans le cadre du contrôle officiel et de l'acceptation internationale des résultats obtenus avec la méthode alternative.
L'ISO 16140:2003 établit également les principes généraux régissant la certification de ces méthodes alternatives, sur la base du protocole de validation défini dans le présent document.
Lorsqu'une méthode alternative est utilisée de façon routinière en laboratoire sans qu'il soit nécessaire de répondre à des critères extérieurs (plus exigeants) en matière d'assurance qualité, une validation comparative de la méthode alternative moins restrictive que ce que prévoit l'ISO 16140:2003 peut convenir.

Mikrobiologija živil in krme - Protokol za validacijo alternativnih metod (ISO 16140:2003)

Ta EN ISO 16140 določa splošni princip in tehnični protokol za validacijo alternativnih metod na področju mikrobiološke analize živil, krme ter okoljskih in veterinarskih vzorcev (glej 5.1.1.2.1) za:
- validacijo alternativnih metod, ki se lahko uporablja predvsem v okviru uradnih kontrol;
- mednarodno sprejemljivost rezultatov, pridobljenih z alternativno metodo.
Prav tako podaja splošna načela certifikacije teh alternativnih metod, zasnovanih na validacijskem protokolu, določenem v tem EN ISO 16140 (glej 4.2).
Kjer se na ustaljen način za interno uporabo v laboratoriju uporablja alternativna metoda brez zahtev za izpolnitev (višjih) zunanjih meril zagotavljanja kakovosti, je lahko primerna manj strožja primerljiva validacija alternativne metode, kot je tista, podana v tem standardu.

General Information

• Status: Withdrawn
• Publication Date: 30-Apr-2003
• Withdrawal Date: 19-Jul-2016
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 6 - Microbiological contamination
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Completion Date: 20-Jul-2016
• Ref Project: SIST EN ISO 16140:2003 - Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods (ISO 16140:2003)

Relations

• Replaced By: EN ISO 16140-1:2016 - Microbiology of the food chain - Method validation - Part 1: Vocabulary (ISO 16140-1:2016)
• Effective Date: 08-Jun-2022
• Replaced By: EN ISO 16140-2:2016 - Microbiology of the food chain - Method validation - Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method (ISO 16140-2:2016)
• Effective Date: 08-Jun-2022
• Amended By: EN ISO 16140:2003/A1:2011 - Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods - Amendment 1 (ISO 16140:2003/AMD 1:2011)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2003
Mikrobiologija živil in krme - Protokol za validacijo alternativnih metod (ISO
16140:2003)
Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative
methods (ISO 16140:2003)
Mikrobiologie von Lebensmitteln und Futtermitteln - Arbeitsvorschrift für die Validierung
alternativer Verfahren (ISO 16140:2003)
Microbiologie des aliments - Protocole pour la validation des méthodes alternatives (ISO
16140:2003)
Ta slovenski standard je istoveten z: EN ISO 16140:2003
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 16140
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2003
ICS 07.100.30
English version
Microbiology of food and animal feeding stuffs - Protocol for the
validation of alternative methods (ISO 16140:2003)
Microbiologie des aliments - Protocole pour la validation Mikrobiologie von Lebensmitteln und Futtermitteln -
des méthodes alternatives (ISO 16140:2003) Arbeitsvorschrift für die Validierung alternativer Verfahren
(ISO 16140:2003)
This European Standard was approved by CEN on 6 July 2002.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2003 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140:2003 E
worldwide for CEN national Members.

Contents
page
Foreword. 3
Introduction . 4
1 Scope. 5
2 Normative references . 5
3 Terms and definitions. 6
4 General principles for the validation and the certification of alternative methods. 7
5 Qualitative methods - Technical protocol for their validation.8
6 Quantitative methods -Technical protocol for their validation . 18
Annex A (normative)  Specific rules for the acceptance of external results already obtained in a prior
validation scheme . 33
Annex B (informative)  Classification of sample types for validation studies . 35
Annex C (normative)  Use of naturally contaminated samples and preparation of artificially
contaminated samples in validation studies. 38
Annex D (normative)  Duplication of samples for the determination of relative accuracy and of relative
detection level for qualitative methods . 40
Annex E (normative)  Calculation of the confidence intervals associated with the number of samples
tested. 42
Annex F (normative)  Test applied to the examination of discordant results. 43
Annex G (normative)  Points to be considered when selecting strains for testing selectivity . 44
Annex H (normative)  Guidelines for the organisation and conducting collaborative studies. 46
Annex I (normative)  Determination that negative controls are free of target analyte. 49
Annex J (normative)  Replication of samples for interlaboratory studies of qualitative methods . 50
Annex K (normative)  Consideration of data .52
Annex L (informative)  Interlaboratory study of qualitative methods: criteria of accordance,
concordance and concordance odds ratio . 53
Annex M (normative)  Replication of samples for the determination of relative accuracy of quantitative
methods . 58
Annex N (normative)  Examples of acceptable and unacceptable situations and range of
measurements for the estimation of the regression line for quantitative methods. 60
Annex O (normative)  Assessment of the linearity of quantitative methods by graphical representation. 62
Annex P (normative)  Detection and quantification limits for counts. 63
Annex Q (normative) Robust estimator of dispersion based on the recursive median Sn from
Rousseeuw [6]. 65
Annex R (normative)  Calculations with the regression method. 66
Annex S (normative)  Examples of calculations for quantitative methods . 71
Annex T (normative)  Collaborative study – Ring test results with duplicates. 76
Annex U (informative)  List of symbols and abbreviations . 77
Bibliography . 78
Foreword
This document (EN ISO 16140:2003) has been prepared by Technical Committee CEN/TC 275 "Food analysis -
Horizontal methods", the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC 34
"Agricultural food products".
This European Standard shall be given the status of a national standard, either by publication of an identical text or
by endorsement, at the latest by November 2003, and conflicting national standards shall be withdrawn at the latest
by November 2003.
The annexes A, C to K and M to T are normative. The annexes B, L and U are informative.
This document contains also a Bibliography.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following coun-
tries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal,
Slovakia, Spain, Sweden, Switzerland and the United Kingdom.
Introduction
The need for the food industry to rapidly assess the microbiological quality of raw materials and finished products
and the microbiological status of manufacturing procedures, has led to the development and refinement of alterna-
tive microbiological methods of analysis that are quicker and/or easier to perform than the corresponding reference
method; some can also be automated.
Among these alternative methods, some can yield results that are equivalent to those provided by the reference
method, while others can lead to results that differ appreciably.
The suppliers/producers of the alternative methods, the food and drink industry, the public health services and
other authorities need a reliable common protocol for the validation of such alternative methods. The data gener-
ated can also be the basis for the certification of a method by an independent organisation.
Because of the extent of the methods comparative study described in this standard for use by the organising labo-
ratory, the procedure is sometimes not appropriate for use as an "in house" method for the validation of an alterna-
tive method by an individual laboratory.
1 Scope
This document establishes the general principle and the technical protocol for the validation of alter-
native methods in the field of microbiological analysis of food, animal feeding stuff and environmental
and veterinary samples (see 5.1.1.2.1) for:
 the validation of alternative methods which can be used in particular in the framework of the offi-
cial control;
 the international acceptance of the results obtained by the alternative method.
It also establishes the general principles of certification of these alternative methods, based on the
validation protocol defined in this EN ISO 16140 (see 4.2).
Where an alternative method is used on a routine basis for internal laboratory use without the re-
quirement to meet (higher) external criteria of quality assurance, a less stringent comparative valida-
tion of the alternative method than that set in this standard may be appropriate.
2 Normative references
This European Standard incorporates by dated or undated reference, provisions from other publica-
tions. These normative references are cited at the appropriate places in the text, and the publications
are listed hereafter. For dated references, subsequent amendments to or revisions of any of these
publications apply to this European Standard only when incorporated in it by amendment or revision.
For undated references the latest edition of the publication referred to applies (including amend-
ments).
ISO 3534-1, Statistics – Vocabulary and symbols – Part 1: Probability and general statistical terms.
ISO 5725, Accuracy (trueness and precision) of measurement methods and results.
3 Terms and definitions
For the purposes of this European Standard, the following terms and definitions apply:
3.1
alternative method
method of analysis that demonstrates or estimates, for a given category of products, the same analyte
(3.4) as is measured using the corresponding reference method (3.2).
NOTE 1 The method can be proprietary or non commercial, and does not need to cover an entire analysis pro-
cedure, that is from the preparation of samples to the test report.
NOTE 2 The alternative method exhibits attributes appropriate to the users' needs, for example:
 speed of analysis and/or response;
 ease of execution and/or automation;
 analytical properties (precision, accuracy, limit of detection, etc.);
 miniaturisation;
 reduction of cost.
NOTE 3 The term "alternative" is used to refer to the entire "test procedure and reaction system". This term
includes all ingredients whether material or otherwise, required for implementing the method.
3.2
reference method
internationally recognised method and widely accepted.
NOTE For the purpose of this standard, these are International and European Standards and if not existing,
certain national standards of equivalent standing.
3.3
validation of an alternative method
demonstration that adequate confidence is provided that the results obtained by the alternative
method are comparable to those obtained using the reference method
NOTE The word "comparable" is defined in this EN ISO 16140 by a technical protocol adapted to each type
of method (see clauses 5 and 6).
3.4
analyte
component measured by the method of analysis. It may be the microorganism
3.5
qualitative method
method of analysis whose response is either the presence or absence of the analyte (3.4) detected
either directly or indirectly in a certain amount of sample
3.6
quantitative method
method of analysis whose response is the amount of the analyte (3.4) measured either directly (enu-
meration in a mass or a volume), or indirectly (colour absorbance, impedance, etc.) in a certain
amount of sample
3.7
methods comparison study
study, performed by the organising laboratory of the alternative method against the reference method
3.8
inter-laboratory study
study of the method’s performance using common samples in several laboratories and under the con-
trol of the organising laboratory
3.9
organising laboratory
laboratory having the qualified staff and skills to perform the method comparison study and organise
the interlaboratory study.
NOTE The availability of an experienced statistician is essential for the analysis of the results.
4 General principles for the validation and the certification of alternative
methods
4.1 Validation protocol
The validation protocol comprises two phases:
 a methods comparison study (3.7) of the alternative method (3.1) against the reference method
(3.2) carried out in the organizin
...